Calcium/NFAT signalling promotes early nephrogenesis.

A number of Wnt genes are expressed during, and are known to be essential for, early kidney development. It is typically assumed that their products will act through the canonical ß-catenin signalling pathway. We have found evidence that suggests canonical Wnt signalling is not active in the early nephrogenic metanephric mesenchyme, but instead provide expressional and functional evidence that implicates the non-canonical Calcium/NFAT Wnt signalling pathway in nephrogenesis. Members of the NFAT (Nuclear Factor Activated in T cells) transcription factor gene family are expressed throughout murine kidney morphogenesis and NFATc3 is localised to the developing nephrons. Treatment of kidney rudiments with Cyclosporin A (CSA), an inhibitor of Calcium/NFAT signalling, decreases nephron formation--a phenotype similar to that in Wnt4(-/-) embryos. Treatment of Wnt4(-/-) kidneys with Ionomycin, an activator of the pathway, partially rescues the phenotype. We propose that the non-canonical Calcium/NFAT Wnt signalling pathway plays an important role in early mammalian renal development and is required for complete MET during nephrogenesis, potentially acting downstream of Wnt4.

K-ras 4A and 4B are co-expressed widely in human tissues, and their ratio is altered in sporadic colorectal cancer.

Ras activating mutations result in constitutive activation of Ras signalling pathways and occur in 30% of human malignancies. K-ras encodes two splice variants, K-ras 4A and 4B, and K-ras activating mutations which jointly affect both isoforms are prevalent in lung, pancreatic and colorectal cancers. Using RT-PCR we examined their expression in normal adult human tissues and addressed whether K-ras splicing is altered in sporadic colorectal cancer by comparing normal colon with colon carcinoma cell lines, and 'matched' tumour and tumour-free colon tissues from the same patient. K-ras 4B was expressed ubiquitously and was the predominant splice variant. K-ras 4A was expressed differentially, with detection in colorectal tumours and cell lines, and normal colon, pancreas and lung--sites where tumours with K-ras activating mutations arise. Both K-ras splice variants were co-expressed by single colon carcinoma cells. The K-ras 4A/4B ratio was significantly reduced in all 6 cell lines examined, including two that lacked K-ras activating mutations, and in 4/9 primary adenocarcinomas. We conclude that K-ras activating mutations do not affect K-ras splicing per se, both isoforms may play a role in neoplastic progression, and altered splicing of either the K-ras proto-oncogene or oncogene, in favour of K-ras 4B, may modulate tumour development.

Gonadal effects of a mouse Denys-Drash syndrome mutation.

Gonadal effects of the Denys-Drash syndrome (DDS) mutation Wt1(tmT396 )were examined in chimaeric and heterozygous mice. Since the only heterozygote was 41,XXY, Sertoli cell function was assessed by comparison with age-matched control XXY testes. Control XXY Sertoli cells showed immuno-expression of WT1 and androgen receptor (AR) indistinguishable from wild-type (40,XY), but expressed anti-Mullerian hormone (AMH). In contrast, DDS Sertoli cells showed only faint immuno-expression of WT1 and did not express AR or AMH. While XY<-->XY DDS chimaeras were male, XX<-->XY chimaeras were predominantly female. In the rare XX<-->XY DDS males the Sertoli cell lineage was largely derived from Wt1 mutant XY cells. We conclude that DDS mutant cells can form Sertoli cells, that the dominant mutation does not cause male sex reversal in mice but distorts the sex ratio of XX<-->XY chimaeras, and that there may be a link between WT1, AMH and AR expression by Sertoli cells in vivo.

Aedes (Finlaya) japonicus (Diptera: Culicidae), a newly recognized mosquito in the United States: analyses of genetic variation in the United States and putative source populations.

Introduction of potential disease vectors into a new geographic area poses health risks to local human, livestock, and wildlife populations. It is therefore important to gain understanding of the dynamics of these invasions, in particular its sources, modes of spread after the introduction, and vectorial potential. We studied the population genetics of Aedes (Finlaya) japonicus japonicus (Theobald), an Asian mosquito that was recognized for the first time in the United States in 1998. We examined patterns of genetic diversity using random amplified polymorphic DNA and sequences of ND4 of mtDNA by comparing samples from populations spanning the range of this mosquito in Japan (six samples) and the United States (nine samples) as well as specimens intercepted in New Zealand in 1999. We found geographically differentiated populations in Japan, indicating limited gene flow even on small spatial scales. In the United States, we found evidence of significant genetic differentiation between samples from New York, Connecticut, and New Jersey and those from mid-Pennsylvania and Maryland. We were unable to pinpoint the source location(s) in Japan, although some of the U.S. samples are genetically close to samples from south Honshu and western Kyushu. Further studies should include samples from Korean populations. Distinct genetic signatures in U.S. populations undergoing expansion suggest the possibility of local increases in genetic diversity if and where they meet.

Platelet gel: an autologous alternative to fibrin glue with applications in oral and maxillofacial surgery.

The preparation and use of platelet gel, an autologous formulation of fibrin glue, are described. The unique features of this biologic sealant are that it is derived from autologous blood collected in the immediate preoperative period by the anesthesiologist, it contains a high concentration of platelets, and it can be used in patients who are not candidates for blood bank donation. Platelet gel has been used successfully in the area of reconstructive oral and maxillofacial surgery in conjunction with ablative surgery of the maxillofacial region, mandibular reconstruction, surgical repair of alveolar clefts and associated oral-antral/ oral-nasal fistulas, and adjunctive procedures related to the placement of osseointegrated implants.

Electrophysiological analysis of dorsal root ganglion neurons pre- and post-coexpression of green fluorescent protein and functional 5-HT3 receptor.

Aequorea green fluorescent protein (GFP) is an excellent marker to examine genetically altered live cells in whole animals or culture. Its potential use in identifying genetically modified neurons, however, has not been investigated extensively. To examine the usefulness, toxicity, and potential electrophyiological effects of GFP expression in neurons, we generated adenovirus containing the mGFP4 cDNA. One week after virus transfection of dorsal root ganglion neurons (DRG), 10% of postnatal DRG neurons appeared brightly fluorescent, labelling the soma and neurites. Temporal examination of these neurons demonstrated no toxicity to DRG neurons even after several weeks in culture with repeated daily epifluorescent exposure. Electrophysiological analysis and comparison of control and viral exposed (GFP- and GFP+) DRG neurons did not demonstrate any differences in whole cell resistance, resting potential, action potential (AP) threshold, AP duration, AP amplitude, or whole cell capacitance. To investigate the usefulness of GFP as a marker for identifying neurons genetically altered to express a novel neurotransmitter receptor, a second adenovirus construct was generated containing both GFP and serotonin type 3 (5-HT3) receptor cDNAs. Transfection of DRG neurons with this virus produced an inward current in the presence of serotonin only in DRG neurons that were GFP-positive. It is concluded that adenoviral transfection of neurons with GFP, for cellular labeling, and coexpression of GFP-neurotransmitter constructs are safe, nontoxic, methods for electrophysiologically investigating neurons over several weeks. The uniqueness of the vector used in these experiments is that it was constructed to express GFP in a second cassette so that it would label the transduced cells, but have no potential for interfering with the function of the foreign 5-HT3 receptor.

Amygdala-kindled seizures increase the expression of corticotropin-releasing factor (CRF) and CRF-binding protein in GABAergic interneurons of the dentate hilus.

Kindling, a model of temporal lobe epilepsy, induces a number of neuropeptides including corticotropin-releasing factor (CRF). CRF itself can produce limbic seizures which resemble kindling in some aspects. However, tolerance to the convulsant effects of CRF develops rapidly. Hypothetically, this could be explained should seizures also induce the CRF-binding protein (CRF-BP), which has been postulated to restrict the actions of CRF. Therefore, in the present study, we used in situ hybridization to examine the effects of amygdala-kindled seizures on the mRNA levels of CRF and CRF-BP. Kindled seizures markedly elevated CRF and CRF-BP in the dentate gyrus of rats. CRF and CRF-BP were induced almost exclusively in GABAergic interneurons of the dentate hilus. The CRF and CRF-BP interneurons also expressed neuropeptide Y but not cholecystokinin. CRF appeared to have an excitatory role in the dentate gyrus as it decreased the afterhyperpolarization of dentate granule neurons. These results suggest that CRF may contribute to the development of amygdala kindling. However, the compensatory induction of CRF-BP may serve to limit the excitatory effects of CRF in the dentate gyrus.

Pyrrolosporin A, a new antitumor antibiotic from Micromonospora sp. C39217-R109-7. I. Taxonomy of producing organism, fermentation and biological activity.

Strain C39217-R109-7 (ATCC 53791) is an actinomycete strain isolated from a soil sample collected at Puerto Viejo, Peru. It produces a new antitumor antibiotic, designated pyrrolosporin A. Taxonomic studies on its morphological, cultural and physiological characteristics identified this producing strain as Micromonospora sp. C39217-R109-7. Pyrrolosporin A shows antimicrobial activity against Gram-positive bacteria and it is weakly active against Gram-negative bacteria. Pyrrolosporin A prolongs the life span of mice inoculated with P388 leukemia cells.

Population size, parity structure, and wing length of Coquillettidia perturbans in an Ohio focus of eastern equine encephalitis.

Adult female density, parity status, and wing length were determined weekly for a population of Coquillettidia perturbans in an area enzootic for eastern equine encephalitis virus in central Ohio. Samples were collected in CO2-baited CDC miniature light traps from the first week in June through the 2nd week of September 1992. Population density indicated a single emergence peak during the 2nd week in July. However, parity rates showed 2 peaks, occurring in the first week of August (70.9% parous) and the 2nd week of September (55.3% parous), which suggested that there was a relatively small 2nd generation. Average wing length declined significantly over the season. The decline in size was negatively correlated with average air temperature occurring at least 6 wk before the time of emergence. Despite the seasonal decline in wing length, the low coefficient of variation for the average wing length (5.5) indicated relatively little variation in size. Comparison of parous and nulliparous female wing lengths each week suggested that there was no association between size and survival in this species.

Rakicidins, new cytotoxic lipopeptides from Micromonospora sp. fermentation, isolation and characterization.

The new cytotoxic agents rakicidins A and B were isolated from cultured broth of a Micromonospora sp. Spectroscopic and amino acid analysis has shown that rakicidin A is a new cyclic lipopeptide, consisting of 4-amino-penta-2,4-dienoic acid, 3-hydroxy-2,4,16-trimethyl-heptadecanoic acid, sarcosine, and 3-hydroxyasparagine. Rakicidin B differs by one methylene group in the lipid side chain. These compounds exhibited cytotoxicity against the M109 cell line.

Korkormicins, novel depsipeptide antitumor antibiotics from Micromonospora sp C39500: fermentation, precursor directed biosynthesis and biological activities.

Micromonospora sp C39500, isolated in our laboratory from a soil sample, produced a complex of seven novel depsipeptide antitumor antibiotics, designated korkormicins. The major component of the complex, korkormicin A, has a MW of 1452 and a molecular formula of C66H84N16O22. Korkormicin A exhibits potent in vivo antitumor activity against P388 leukemia and M109 lung carcinoma implanted intraperitoneally (ip) in mice, with effective doses of 0.05-0.20 mg kg-1 injection-1, for five or three ip injections, respectively. It is also active against Gram-positive bacteria but inactive against Gram-negative bacteria. The production of korkormicin A was enhanced by 3-fold when 0.1% L-valine was added to the production culture at 48 h. A titer of 401.0 micrograms ml-1 was achieved in the fermenter culture supplemented with 0.1% L-valine.

Effect of mechanical shock on hydrostatic balance and survival of mosquito pupae.

The presence and distribution of ventral air space gas assures that mosquito pupae are positively buoyant and that they float, dive, and ascend in an upright, balanced orientation. Our objective was to test the effects of mechanical shocks of varying magnitude on mosquito pupae representing 3 genera. Forces that disrupt the pupa's buoyancy and/or hydrostatic balance are of a much lower magnitude than those that would cause tissue damage. Once hydrostatic balance and/or buoyancy are compromised, pupae are unable to restore them and eventually drown. This could represent a weak link in the mosquito life cycle. It is possible that mechanical shock or sound-generating devices could be designed that could break this link.

Visual deprivation decreases long-term potentiation in rat visual cortical slices.

A major finding in the visual plasticity literature is that visual deprivation is effective only during an early 'sensitive' period, which is lengthened by dark rearing. Unresolved is whether the visual cortex is in a normally plastic state prior to light stimulation. This cannot be addressed using paradigms employing light exposure to assess plasticity. Several developmental studies have investigated a plastic phenomenon termed long-term potentiation (LTP) in slices from cat (J. Neurophysiol., 59 (1988) 124-141) and rat (Brain Res., 439 (1988) 222-229) visual cortex. Susceptibility to the induction of LTP parallels the period of sensitivity to visual deprivation. This suggests that slices can be used to assay visual cortical plasticity, avoiding light exposure. In the present study, field potentials were recorded from slices of the primary visual cortices of dark-reared (DR) and control (CONT) Long Evans hooded rats (17 to 21 days). Field potential profiles recorded before and 90 min following tetanic electrical stimulation were subjected to current source density analysis, yielding extracellular current sink amplitudes. Tetanus resulted in LTP in both CONT and DR slices, but DR slices were significantly less potentiated. These results indicate that the primary visual cortex of DR animals is not fully plastic, indicating a role for light stimulation in inducing visual cortical plasticity.

Records of mosquito-parasitic mermithid nematodes in the northcentral United States.

Mermithid parasites (Nematoda: Mermithidae) were observed in 11 mosquito species in 22 counties of Indiana, Michigan, Minnesota, Ohio and Wisconsin. Natural hosts included adult Aedes vexans, Ae. canadensis, Ae. cinereus, Ae. diantaeus, Ae. punctor, Anopheles punctipennis and Coquillettidia perturbans and larvae of Ae. abserratus, Ae. canadensis, Ae. cinereus, Ae. communis, Ae. diantaeus, Ae. provocans, Ae. punctor, Ae. stimulans and Cq. perturbans. These are the first records of such parasites in Indiana, Minnesota, Ohio and Wisconsin.

Eupenifeldin, a novel cytotoxic bistropolone from Eupenicillium brefeldianum.

Eupenifeldin was isolated from cultures of Eupenicillium brefeldianum ATCC 74184 by extraction and crystallization. The compound was identified as a pentacyclic bistropolone on the basis of spectral data and its complete structure was established by single-crystal X-ray analysis. The compound is cytotoxic against the HCT-116 cell line and has in vivo antitumor activity in the P388 leukemia model.

Eastern equine encephalitis virus in Ohio during 1991.

During August and September of 1991, an epizootic of eastern equine encephalitis (EEE) virus in horses occurred in Wayne and Holmes countries, OH. This was the first recorded epizootic of EEE virus in the state. Twelve horses were confirmed positive for EEE virus through virus isolation or seroconversion, and seven additional horses with compatible symptoms were in close spatial and temporal proximity to the confirmed cases and were presumed to have died from EEE virus. The outbreak was centered around the Killbuck Wildlife Area, a 2,147-ha tract maintained by the state, half of which consists of wooded swamp and marsh. Mosquitoes were collected in upland areas before the epizootic and in the swamp basin at the end of the epizootic to identify the mosquito species involved in EEE virus transmission. We collected and tested 22,095 specimens for the presence of virus. EEE virus was isolated from one pool of the most likely epizootic vector, Coquillettidia perturbans (Walker). The minimum infection rate for EEE virus in this species was 0.1/1,000. Dense populations of Aedes vexans (Meigen) and Culex salinarius Coquillett occurred in the area, but their densities peaked after the epizootic. It is unlikely that these species were involved in epizootic transmission. IgM antibody to EEE virus was detected in three bird species collected in the swamp.

Effect of pH of bupivacaine on duration of repeated sciatic nerve blocks in the albino rat. Local Anesthetics for Neuralgia Study Group.

Tachyphylaxis has been ascribed to tissue acidification after repeated injections of acidic local anesthetic solutions. We studied the effect of pH on the duration of action of bupivacaine to determine the validity of this proposed mechanism of tachyphylaxis by injecting bupivacaine solutions adjusted to pH 4.2 or 6.8 into a surgically implanted system created to permit in vivo irrigation of rat sciatic nerves with local anesthetic. Tachyphylaxis developed at both pH values. The results fail to support the acidification hypothesis as there was no statistically significant effect of a 400-fold difference in hydrogen ion concentration on the development of tachyphylaxis or the duration of motor dysfunction.

A slice preparation preserving the callosal projection to contralateral visual cortex.

Due to the curved path they follow, the visual callosal projections to areas OC1 and OC2 of the rat visual cortex have been inaccessible to studies using brain slices. In this paper we describe a new slice preparation in which a curved cutting blade was used to obtain slices in which callosal fibers projecting to OC1 or OC2 are preserved. Stimulation of the contralateral white matter resulted in EPSPs recorded in layer II/III and V cells of OC2 studied with intracellular recording. Current source density analysis of extracellular field potentials collected in OC1 and OC2 revealed laminar current sink patterns paralleling the laminar distribution of callosal terminations reported by Miller and Vogt (Dev. Brain Res., 14 (1984) 304-309). Exposure of slices to 2 mM kynurenic acid reversibly abolished current sinks in OC1 recorded in response to callosal stimulation indicating that glutamate receptors mediate the response of OC1 to callosal afferent activity. This new slicing technique can be readily adapted to study other systems in the nervous system in which neural processes follow curved trajectories.